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Introduction Heart rate variability (HRV) is regarded as a useful, non-invasive method for investigating the autonomic nervous system (ANS). Over the past decade there has been an increase in the number of HRV investigations in the disciplines of medical, sport and exercise science. Despite the extensive use of HRV in investigations of ANS functioning, there are questions relating to the reliability of the technique. Therefore, HRV reliability studies for different population groups have been advocated. Furthermore, research on gender differences in HRV is contradictory. This has resulted in the need to investigate gender characteristics in HRV. Objectives The objective of this study was to evaluate the reliability of short-term (5min) recordings of HRV, and to determine the association between HRV and gender. Methods Forty four physically active students (n= 21, age= 21.17 (1.55) males and n=23, age= 19.75 (1.76) females) participated in the study. Heart rate variability parameters were determined from five minute recording of interbeat intervals (IBI) using a Suunto t6 heart rate monitor (HRM). Testing was repeated over 4 consecutive days under the same conditions. The following HRV time and frequency domain measures were calculated using Kubios HRV Software Version 2.0: mean heart rate (HR), standard deviation of normal to normal intervals (SDNN), root mean square of successive differences (RMSSD), percentage of beats that changed more than 50 ms from the previous beat (pNN50), low frequency in normalized units (LFnu), high frequency in normalized units (HFnu) and low frequency to high frequency ratio in normalized units (LF/HFnu ratio). The data was summarized using routine descriptive statistics. Relative reliability was calculated using interclass correlation coefficients (ICC) (ICC of >0.80 indicated good to excellent reliability) and absolute reliability using typical error of measurement (TEM) and TEM as a percentage of the mean score (TEM%). This statistical measures were computed for days 2 vs 3 (REL 1), 3 vs 4 (REL 2). Day 1 was used as a familiarization day. An unpaired T-test was used to determine whether there were any differences between males and females for the above HRV parameters. Significance was set at p .0.05. Results The ICCs for both REL 1 and REL 2 indicated good to excellent (ICC >0.8) reliability for IBIs and pNN50 for the time domain results. In general, the time domain results had a higher relative reliability than the frequency domain results. Males had an overall lower relative reliability than females for frequency domain parameters. Absolute reliability for REL 2 showed a slightly lower TEM value as compared to REL 1.The largest gender differences in TEM were seen in the frequency domain parameters. Specifically, for males, the TEM was higher than females for the LF/HFnu ratio (REL 2: 116%), the HFnu (REL 1: 90%) and the LFnu (REL 1: 68%). Overall the TEM% was relatively high in most HRV parameters specifically for LF/HFnu (REL 1: 31.4% females and 48.1% males; REL 2: 29.7% females and 40.4% males). These findings indicate that males have decreased absolute reliability compared to females and that random error is greater in men for the frequency domain parameters. Gender differences illustrated significant differences for resting HR (16% higher in females (p < 0.0001)), IBIs (21% higher in females (p <0.0001)) and LF/HFnu ratio (41% higher in males (p = 0.003)). The findings indicate that females have higher total HRV. Conclusions Short term recordings of HRV over consecutive days using the Suunto t6 HRM and Kubios custom HRV software are reliable depending on the HRV parameter being analysed. Overall, the relative reliability results suggest that HRV using the Suunto t6 and Kubios is good. However, the absolute reliability results suggest low reliability. In particular, males demonstrated a poorer absolute reliability (high TEM and TEM%) than females, suggesting a larger day to day random error in males. Furthermore, specific HRV measures differed between males and females demonstrating that females have higher parasympathetic modulation compared to men. The overall higher HRV in females could explain the possible cardio-protective mechanism observed in premenopausal women. Key words: Heart rate variability, Parasympathetic, Reliability, Interbeat Intervals / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.